Bacterial diseases could severely harm agricultural production. To develop new antibacterial agents, the secondary metabolites of a deep-sea-derived fungus Simplicillium obclavatumEIODSF 020 with antibacterial activities against plant and fish pathogens were investigated by a bioassay-guided approach, which led to the isolation of 11 new peptaibiotics, simplicpeptaibs A–K (1–11). They contain 16–19 residues, including β-alanine, tyrosine, or tyrosine O-sulfate, that were rarely present in peptaibiotics. Their structures were elucidated by spectroscopic analyses (NMR, HRMS, HRMS2, and ECD) and Marfey’s method. The primary and secondary structures of novel sulfated peptaibiotic 9were reconfirmed by single-crystal X-ray diffraction analysis. Genome sequencing of S. obclavatumEIODSF 020 allowed the detection of a gene cluster encoding two individual NRPSs (totally containing 19 modules) that was closely related to simplicpeptaib biosynthesis. Antibacterial investigations of 1–11together with the previously isolated linear and cyclic peptides from this strain suggested the antibacterial property of this fungus was attributed to the peptaibiotics and cyclic lipopeptides. Among them, compounds 4, 6, 7, and 9showed significant activity against the tobacco pathogen Ralstonia solanacearumor tilapia pathogens Streptococcus iniaeand Streptococcus agalactiae. The antibacterial activity of 6against R. solanacearumcould be enhanced by the addition of 1% NaCl. The structure–bioactivity relationship of simplicpeptaibs was discussed.